Fungal infections are a serious public-health hazard, particularly for the growing population of immunocompromised patients, including those with AIDS and organ transplant recipients. A rise in fungal infections is also seen in elderly people, denture wearers and individuals with salivary dysfunction. Many presently available antifungal agents are toxic and/or not effective against an increasing number of the drug-resistant fungal strains. Human salivary histatins (Hsns) are a group of cationic peptides that have been shown to possess fungicidal activity. This property coupled with the fact that Hsns are natural products of the human body and thus most likely nontoxic makes them promising antifungal therapeutic agents. In particular, the ability of histatins to kill azole-resistant strains of C. albicans and Cryptococcus neoformans indicates that they may have therapeutic potential in treating fungal infections associated with AIDS. During the previous grant period, we examined the structure- function relationship of Hsn-5 (24 amino acid residue protein) with respect to its candidacidal activity using recombinant Hsn-5 variants as well as chemically synthesized Hsn-5 fragments. In this proposal, we will explore the hypotheses that Hsns can be effective natural antifungal therapeutic agents and that Hsn variants with enhanced protective functions can be designed if the Hsn structure-function relationship (Specific Aim 1) and its mode of action on C. albicans (Specific Aim 2) are better defined and understood. Based on the outcomes from the first two specific aims and our previous results, we will design Hsn-5 variants with enhanced activity and/or stability and examine them for their potential suitability as antifungal therapeutic agents both in vitro and in vivo (Specific Aim 3). We will also examine whether Hsn-5 and relevant variants possess fungicidal activity against other opportunistic fungal species, and act synergistically with other antifungal agents (Specific Aim 4). The long-term objective of this research is to design and produce novel Hsns with enhanced protective function that may serve as effective and non-toxic natural antifungal therapeutic agents that would help to outpace the growing list of the drug resistant and opportunistic fungi causing life-threatening, disseminated diseases. The same molecules could also be used as components of artificial saliva for patients with salivary dysfunction. Collectively, the information obtained can be used to effectively design Hsn-based therapeutic delivery systems for future clinical use.